Cui Xiao-Juan, Lu Zhuo, Xiao Si-Meng, Fang Xue-Wan, Wen Yi-Lei, Xiong Wan-Na, Yu Kai, Liang Jian-Qin
Guangxi University of Chinese Medicine Nanning 530200, China.
the First Affiliated Hospital of Guangxi University of Chinese Medicine Nanning 530023, China.
Zhongguo Zhong Yao Za Zhi. 2021 Jan;46(1):206-213. doi: 10.19540/j.cnki.cjcmm.20200618.201.
This paper was to investigate the effect of Huanglian Jiedu Decoction(HLJD) on ulcerative colitis(UC) in mice, and determine the effective components in plasma, and virtually screen its therapeutic target, and predict its mechanism. Sixty Balb/c mice were randomly divided into blank group, model group, mesalazine treatment group(0.3 g·kg(-1)), and HLJD treatment groups(24.66, 12.33, 6.17 g·kg(-1)). Excepted for the blank group, all the mice in HLJD and mesalazine treatment groups were gavage administration. All mice freely drank 2.5% DSS solution for seven days to induce UC. The disease activity index(DAI) was detected each day. At the end of the experiment, HE staining was used to observe the pathological changes in colon. The content of IL-1β, IL-6 and TNF-α in colon were determined by ELISA. The effective components in plasma were determined by UPLC-Q-TOF-MS. The reverse docking in PharmMapper was used to screen the component targets. The disease targets of UC were collected by searching TTD, OMIM and GeneCards databases. The intersection of the component targets and disease targets was selected as the therapeutic targets. Then the therapeutic targets were imported into the STRING for GO and KEGG enrichment analysis. Discovery Studio was used to simulate the docking between the components and the targets. RESULTS:: showed that the DAI in the model group increased significantly(P<0.05), and the number of inflammatory cells and infiltration degree increased significantly compared with the blank group. The DAI in HLJD treatment group was significantly reduced(P<0.05), and the number and infiltration degree of inflammatory cells were reduced compared with the model group. The ELISA results showed that the levels of IL-1β, IL-6 and TNF-α were increased significantly in the model group(P<0.01) compared with the blank group, and significantly down regulated in the HLJD treatment group(P<0.05) compared with the model group. After UPLC-Q-TOF-MS analyse, ten components were identified. The network pharmacology analysis showed that the action targets were significantly enriched in 129 of biological processes, such as response to organic substance, chemical and oxygen-containing compound, etc., as well as 16 of signal pathways, such as IL-17, TNF and hepatitis B signal pathways, were enriched too. The results of molecular docking showed that limonin, palmatine and berberine could bind to CASP3 and MMP9 by hydrogen bond. In conclusion, HLJD could alleviate the colonic mucosal inflammatory infiltration and mucosal damage in UC mice. The mechanism may be related to the anti-inflammatory effect on UC mice by reducing the levels of IL-1β, IL-6 and TNF-α in colon through limonin, palmatine and berberine regulating IL-17 signal pathway and TNF signal pathway via CASP3 and MMP9 meditated.
本文旨在研究黄连解毒汤(HLJD)对小鼠溃疡性结肠炎(UC)的影响,确定血浆中的有效成分,虚拟筛选其治疗靶点,并预测其作用机制。将60只Balb/c小鼠随机分为空白组、模型组、美沙拉嗪治疗组(0.3 g·kg⁻¹)和HLJD治疗组(24.66、12.33、6.17 g·kg⁻¹)。除空白组外,HLJD治疗组和美沙拉嗪治疗组的小鼠均进行灌胃给药。所有小鼠自由饮用2.5%的葡聚糖硫酸钠(DSS)溶液7天以诱导UC。每天检测疾病活动指数(DAI)。实验结束时,采用苏木精-伊红(HE)染色观察结肠的病理变化。通过酶联免疫吸附测定(ELISA)法测定结肠中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的含量。采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)法测定血浆中的有效成分。利用PharmMapper中的反向对接筛选成分靶点。通过搜索治疗靶点数据库(TTD)、在线人类孟德尔遗传数据库(OMIM)和基因卡片数据库(GeneCards)收集UC的疾病靶点。选择成分靶点和疾病靶点的交集作为治疗靶点。然后将治疗靶点导入STRING进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。利用发现工作室(Discovery Studio)模拟成分与靶点之间的对接。结果显示,模型组的DAI显著升高(P<0.05),与空白组相比,炎症细胞数量和浸润程度显著增加。HLJD治疗组的DAI显著降低(P<0.05),与模型组相比,炎症细胞数量和浸润程度减少。ELISA结果显示,与空白组相比,模型组中IL-1β、IL-6和TNF-α的水平显著升高(P<0.01),与模型组相比,HLJD治疗组中这些指标显著下调(P<0.05)。经过UPLC-Q-TOF-MS分析,鉴定出10种成分。网络药理学分析表明,作用靶点在129个生物学过程中显著富集,如对有机物质、化学物质和含氧化合物的反应等,同时也在16条信号通路中富集,如IL-17、TNF和乙型肝炎信号通路等。分子对接结果显示,柠檬苦素、巴马汀和小檗碱可通过氢键与半胱天冬酶-3(CASP3)和基质金属蛋白酶-9(MMP9)结合。综上所述,HLJD可减轻UC小鼠结肠黏膜的炎症浸润和黏膜损伤。其机制可能与通过柠檬苦素、巴马汀和小檗碱经由CASP3和MMP9介导调节IL-17信号通路和TNF信号通路,降低结肠中IL-1β、IL-6和TNF-α的水平,从而对UC小鼠产生抗炎作用有关。